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BioRxiv : the Preprint Server For... May 2024The bone-resorbing activity of osteoclasts plays a critical role in the life-long remodeling of our bones that is perturbed in many bone loss diseases. Multinucleated...
The bone-resorbing activity of osteoclasts plays a critical role in the life-long remodeling of our bones that is perturbed in many bone loss diseases. Multinucleated osteoclasts are formed by the fusion of precursor cells, and larger cells - generated by an increased number of cell fusion events - have higher resorptive activity. We find that osteoclast fusion and bone-resorption are promoted by reactive oxygen species (ROS) signaling and by an unconventional low molecular weight species of La protein, located at the osteoclast surface. Here, we develop the hypothesis that La's unique regulatory role in osteoclast multinucleation and function is controlled by a ROS switch in La trafficking. Using antibodies that recognize reduced or oxidized species of La, we find that differentiating osteoclasts enrich an oxidized species of La at the cell surface, which is distinct from the reduced La species conventionally localized within cell nuclei. ROS signaling triggers the shift from reduced to oxidized La species, its dephosphorylation and delivery to the surface of osteoclasts, where La promotes multinucleation and resorptive activity. Moreover, intracellular ROS signaling in differentiating osteoclasts oxidizes critical cysteine residues in the C-terminal half of La, producing this unconventional La species that promotes osteoclast fusion. Our findings suggest that redox signaling induces changes in the location and function of La and may represent a promising target for novel skeletal therapies.
PubMed: 38903088
DOI: 10.1101/2024.05.02.592254 -
Frontiers in Endocrinology 2024Gonadotropin-releasing hormone (GnRH) is a key stimulator for gonadotropin secretion in the pituitary and its pivotal role in reproduction is well conserved in...
Differential involvement of cAMP/PKA-, PLC/PKC- and Ca/calmodulin-dependent pathways in GnRH-induced prolactin secretion and gene expression in grass carp pituitary cells.
Gonadotropin-releasing hormone (GnRH) is a key stimulator for gonadotropin secretion in the pituitary and its pivotal role in reproduction is well conserved in vertebrates. In fish models, GnRH can also induce prolactin (PRL) release, but little is known for the corresponding effect on PRL gene expression as well as the post-receptor signalling involved. Using grass carp as a model, the functional role of GnRH and its underlying signal transduction for PRL regulation were examined at the pituitary level. Using laser capture microdissection coupled with RT-PCR, GnRH receptor expression could be located in carp lactotrophs. In primary cell culture prepared from grass carp pituitaries, the native forms of GnRH, GnRH2 and GnRH3, as well as the GnRH agonist [D-Arg, Pro, NEt]-sGnRH were all effective in elevating PRL secretion, PRL mRNA level, PRL cell content and total production. In pituitary cells prepared from the rostral pars distalis, the region in the carp pituitary enriched with lactotrophs, GnRH not only increased cAMP synthesis with parallel CREB phosphorylation and nuclear translocation but also induced a rapid rise in cytosolic Ca by Ca influx via L-type voltage-sensitive Ca channel (VSCC) with subsequent CaM expression and NFAT dephosphorylation. In carp pituitary cells prepared from whole pituitaries, GnRH-induced PRL secretion was reduced/negated by inhibiting cAMP/PKA, PLC/PKC and Ca/CaM/CaMK-II pathways but not the signalling events via IP and CaN/NFAT. The corresponding effect on PRL mRNA expression, however, was blocked by inhibiting cAMP/PKA/CREB/CBP and Ca/CaM/CaN/NFAT signalling but not PLC/IP/PKC pathway. At the pituitary cell level, activation of cAMP/PKA pathway could also induce CaM expression and Ca influx via VSCC with parallel rises in PRL release and gene expression in a Ca/CaM-dependent manner. These findings, as a whole, suggest that the cAMP/PKA-, PLC/PKC- and Ca/CaM-dependent cascades are differentially involved in GnRH-induced PRL secretion and PRL transcript expression in carp lactotrophs. During the process, a functional crosstalk between the cAMP/PKA- and Ca/CaM-dependent pathways may occur with PRL release linked with CaMK-II and PKC activation and PRL gene transcription caused by nuclear action of CREB/CBP and CaN/NFAT signalling.
Topics: Animals; Carps; Gonadotropin-Releasing Hormone; Prolactin; Pituitary Gland; Protein Kinase C; Cyclic AMP-Dependent Protein Kinases; Calcium; Type C Phospholipases; Cyclic AMP; Signal Transduction; Calmodulin; Cells, Cultured; Gene Expression
PubMed: 38894746
DOI: 10.3389/fendo.2024.1399274 -
Journal of Pharmacological Sciences Aug 2024Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT...
Previously, we have shown that pyrogallol alleviated nasal symptoms and suppressed IL-9 gene up-regulation in allergy model rats by inhibiting calcineurin/NFAT signaling. As pyrogallol has antioxidative activity, it may be responsible for inhibiting calcineurin/NFAT signaling-mediated IL-9 gene expression. However, the relationship between antioxidative activity and suppression of IL-9 gene expression has not been elucidated yet. Here, we conducted the structure-activity relationship studies of pyrogallol and its structurally related compounds to understand the mechanism of IL-9 gene suppression by pyrogallol. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay showed that the antioxidative activity of catechol, resorcinol, phloroglucinol, and gallic acid is 60.1%, 10.4%, 18.8%, and 113.5% of pyrogallol, respectively. Catechol, resorcinol, and phloroglucinol did not suppress NFAT dephosphorylation. Gallic acid suppressed dephosphorylation of NFAT. Gallic acid also suppressed ionomycin-induced up-regulation of IL-9 gene expression with the IC value of 82.6 μM. However, catechol, resorcinol and phloroglucinol showed no suppressive activity. In addition, using gallic acid-immobilized beads, we isolated and identified Poly(U)-binding-splicing factor 60 (PUF60) as a pyrogallol binding protein. These results suggest that the antioxidative activity of pyrogallol is not likely to be the mechanism of IL-9 gene suppression. Data also suggest that PUF60 is one of its target molecules responsible for the suppression of calcineurin/NFAT signaling by pyrogallol.
Topics: Pyrogallol; Calcineurin; Signal Transduction; NFATC Transcription Factors; Structure-Activity Relationship; Antioxidants; Humans; Gallic Acid; Gene Expression; Animals; Phosphorylation; Up-Regulation; Rats
PubMed: 38880548
DOI: 10.1016/j.jphs.2024.06.002 -
Nature Communications Jun 2024Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein...
Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C's multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.
Topics: Phosphorylation; Humans; Protein Phosphatase 1; Carrier Proteins; Animals; Protein Phosphatase 2; Myocardium; Protein Kinases; Kinetics
PubMed: 38877002
DOI: 10.1038/s41467-024-49408-5 -
Frontiers in Plant Science 2024Tobacco ( L.) is an important industrial crop, which is sensitive to chilling stress. Tobacco seedlings that have been subjected to chilling stress readily flower early,...
Tobacco ( L.) is an important industrial crop, which is sensitive to chilling stress. Tobacco seedlings that have been subjected to chilling stress readily flower early, which seriously affects the yield and quality of their leaves. Currently, there has been progress in elucidating the molecular mechanisms by which tobacco responds to chilling stress. However, little is known about the phosphorylation that is mediated by chilling. In this study, the transcriptome, proteome and phosphoproteome were analyzed to elucidate the mechanisms of the responses of tobacco shoot and root to chilling stress (4 °C for 24 h). A total of 6,113 differentially expressed genes (DEGs), 153 differentially expressed proteins (DEPs) and 345 differential phosphopeptides were identified in the shoot, and the corresponding numbers in the root were 6,394, 212 and 404, respectively. This study showed that the tobacco seedlings to 24 h of chilling stress primarily responded to this phenomenon by altering their levels of phosphopeptide abundance. Kyoto Encyclopedia of Genes and Genomes analyses revealed that starch and sucrose metabolism and endocytosis were the common pathways in the shoot and root at these levels. In addition, the differential phosphopeptide corresponding proteins were also significantly enriched in the pathways of photosynthesis-antenna proteins and carbon fixation in photosynthetic organisms in the shoot and arginine and proline metabolism, peroxisome and RNA transport in the root. These results suggest that phosphoproteins in these pathways play important roles in the response to chilling stress. Moreover, kinases and transcription factors (TFs) that respond to chilling at the levels of phosphorylation are also crucial for resistance to chilling in tobacco seedlings. The phosphorylation or dephosphorylation of kinases, such as CDPKs and RLKs; and TFs, including VIP1-like, ABI5-like protein 2, TCP7-like, WRKY 6-like, MYC2-like and CAMTA7 among others, may play essential roles in the transduction of tobacco chilling signal and the transcriptional regulation of the genes that respond to chilling stress. Taken together, these findings provide new insights into the molecular mechanisms and regulatory networks of the responses of tobacco to chilling stress.
PubMed: 38872895
DOI: 10.3389/fpls.2024.1390993 -
Frontiers in Cardiovascular Medicine 2024Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca homeostasis and contractility of the heart via dephosphorylation of regulatory...
BACKGROUND
Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca homeostasis and contractility of the heart via dephosphorylation of regulatory proteins. In some genetically modified mouse models with increased arrhythmogenicity, a reduced expression of the regulatory subunit B56α of PP2A was found as a concomitant effect. Whether there is a general correlation between the abundance of B56α and the promotion of cardiac arrhythmogenesis remains unclear.
METHODS
The aim of this study was therefore to investigate the role of PP2A-B56α in the propensity for arrhythmic activity in the heart. The experimental analysis of this question has been addressed by using a mouse model with deletion of the PP2A-B56α gene, (KO), in comparison to wild-type animals (WT). Evidence for arrhythmogenicity was investigated in whole animal, isolated heart and cardiomyocytes by ECG, recording of monophasic action potential (MAP) induced by programmed electrical stimulation (PES), measurement of Ca transients under increased pacing frequencies and determination of total K channel currents ( ).
RESULTS
ECG measurements showed a prolongation of QT time in KO vs. WT. KO mice exhibited a higher rate of premature ventricular contractions in the ECG. MAP measurements in Langendorff-perfused KO hearts showed increased episodes of ventricular tachyarrhythmia induced by PES. However, the KO hearts showed values for MAP duration that were similar to those in WT hearts. In contrast, KO showed more myocardial cells with spontaneous arrhythmogenic Ca transient events compared to WT. The whole-cell patch-clamp technique applied to ventricular cardiomyocytes revealed comparable peak potassium channel current densities between KO and WT.
CONCLUSION
These findings support the assumption that a decrease or even the loss of PP2A-B56α leads to an increased propensity of triggered arrhythmias. This could be based on the increased spontaneous Ca tansients observed.
PubMed: 38863902
DOI: 10.3389/fcvm.2024.1419597 -
Cell Communication and Signaling : CCS Jun 2024Interleukin 33 (IL-33) is a crucial inflammatory factor that functions as an alarm signal in endometriosis (EMs). Epithelial-mesenchymal transition (EMT), a process...
OBJECTIVES
Interleukin 33 (IL-33) is a crucial inflammatory factor that functions as an alarm signal in endometriosis (EMs). Epithelial-mesenchymal transition (EMT), a process related to inflammatory signals, intracellular reactive oxygen species (ROS) production, and lipid peroxidation, have been proposed as potential mechanisms that contribute to the development and progression of EMs. IL-33 is highly upregulated in the ectopic milieu. Moreover, ectopic endometrial cells constitutively express interleukin-33 receptor ST2 (IL-33R). However, the role of IL-33/ST2 in the EMT of EMs remains largely unknown. In this study, we aimed to mechanistically determine the role of IL-33/ST2 in EMs-associated fibrosis.
MATERIALS AND METHODS
We established a non-lethal oxidative stress model to explore the conditions that trigger IL-33 induction. We performed α-smooth muscle actin (α-SMA) protein detection, cell counting kit-8 (CCK-8) assays, and scratch assays to analyze the impact of IL-33 on primary endometrial stromal cells (ESCs) proliferation and invasion. Clinical samples from patients with or without EMs were subjected to immunohistochemical (IHC) and and immunofluorescence(IF) staining to assess the clinical relevance of IL-33 receptor ST2 and EMT-related proteins. Furthermore, we used the ectopic human endometrial epithelial cell line 12Z and normal human epithelial cell line EEC to evaluate the effects of IL-33 on Wnt/β-catenin signaling. The effect of IL-33 on EMT-associated fibrosis was validated in vivo by intraperitoneal injections of IL-33 and antiST2.
RESULTS
We observed that ectopic milieu, characterized by ROS, TGF-β1, and high level of estrogen, triggers the secretion of IL-33 from ectopic ESCs. Ectopic endometrial lesions exhibited higher level of fibrotic characteristics and ST2 expression than that in the normal endometrium. Exogenous recombinant human (rhIL-33) enhanced ESC migration and survival. Similarly, 12Z cells displayed a higher degree of EMT characteristics with elevated expression of CCN4 and Fra-1, downstream target genes of the WNT/β-catenin pathway, than that observed in EECs. Conversely, blocking IL-33 with neutralizing antibodies, knocking down ST2 or β-catenin with siRNA, and β-catenin dephosphorylation abolished its effects on EMT promotion. In vivo validation demonstrated that IL-33 significantly promotes EMs-related fibrosis through the activation of Wnt/β-catenin signaling.
CONCLUSION
Our data strongly support the vital role of the IL-33/ST2 pathway in EMs-associated fibrosis and emphasize the importance of the EMT in the pathophysiology of fibrosis. Targeting the IL-33/ST2/Wnt/β-catenin axis may hold promise as a feasible therapeutic approach for controlling fibrosis in EMs.
Topics: Female; Endometriosis; Interleukin-33; Epithelial-Mesenchymal Transition; Humans; Interleukin-1 Receptor-Like 1 Protein; beta Catenin; Animals; Phosphorylation; Mice; Endometrium; Adult; Cell Proliferation; Cell Movement; Signal Transduction
PubMed: 38858740
DOI: 10.1186/s12964-024-01683-x -
Journal of Molecular and Cellular... Jun 2024Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial...
Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury.
PubMed: 38851627
DOI: 10.1016/j.yjmcc.2024.05.014 -
Cell Death & Disease Jun 2024Integrin αvβ6 holds promise as a therapeutic target for organ fibrosis, yet targeted therapies are hampered by concerns over inflammatory-related side effects. The...
Integrin αvβ6 holds promise as a therapeutic target for organ fibrosis, yet targeted therapies are hampered by concerns over inflammatory-related side effects. The role of αvβ6 in renal inflammation remains unknown, and clarifying this issue is crucial for αvβ6-targeted treatment of chronic kidney disease (CKD). Here, we revealed a remarkable positive correlation between overexpressed αvβ6 in proximal tubule cells (PTCs) and renal inflammation in CKD patients and mouse models. Notably, knockout of αvβ6 not only significantly alleviated renal fibrosis but also reduced inflammatory responses in mice, especially the infiltration of pro-inflammatory macrophages. Furthermore, conditional knockout of αvβ6 in PTCs in vivo and co-culture of PTCs with macrophages in vitro showed that depleting αvβ6 in PTCs suppressed the migration and pro-inflammatory differentiation of macrophages. Screening of macrophage activators showed that αvβ6 in PTCs activates macrophages via secreting IL-34. IL-34 produced by PTCs was significantly diminished by αvβ6 silencing, and reintroduction of IL-34 restored macrophage activities, while anti-IL-34 antibody restrained macrophage activities enhanced by αvβ6 overexpression. Moreover, RNA-sequencing of PTCs and verification experiments demonstrated that silencing αvβ6 in PTCs blocked hypoxia-stimulated IL-34 upregulation and secretion by inhibiting YAP expression, dephosphorylation, and nuclear translocation, which resulted in the activation of Hippo signaling. While application of a YAP agonist effectively recurred IL-34 production by PTCs, enhancing the subsequent macrophage migration and activation. Besides, reduced IL-34 expression and YAP activation were also observed in global or PTCs-specific αvβ6-deficient injured kidneys. Collectively, our research elucidates the pro-inflammatory function and YAP/IL-34/macrophage axis-mediated mechanism of αvβ6 in renal inflammation, providing a solid rationale for the use of αvβ6 inhibition to treat kidney inflammation and fibrosis.
Topics: Animals; Macrophages; Mice; Humans; Integrins; Renal Insufficiency, Chronic; Mice, Knockout; Inflammation; Male; Antigens, Neoplasm; Mice, Inbred C57BL; Signal Transduction; Disease Models, Animal; YAP-Signaling Proteins; Kidney Tubules, Proximal; Fibrosis
PubMed: 38844455
DOI: 10.1038/s41419-024-06785-5 -
Cell May 2024Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At...
Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.
PubMed: 38838667
DOI: 10.1016/j.cell.2024.05.002